基于VIP与Ghrelin信号通路变化探讨运脾开胃颗粒治疗厌食症机理

The project is based on the establishment of the hypotheses that the mechanism of Spleen appetizer Act treating anorexia is on account of the regulation of VIP, Ghrelin signaling pathway. The previous research usually restricted to the initial abnormal expression of signaling molecules and the terminal effectors, our project will overcome these obstacles. We focused on the signaling transduction intermediates which directed at the hot spot of international digestive system research (gastrointestinal peptide receptor, the signal received, information conduction, etc.). By using the advanced technologies (such as Real-Time PCR, LSCM, patch clamp recording technique etc.), we can observe the effects of Spleen appetizer particles (a representative drug of this therapeutic method) on the gastrointestinal hormones such as VIP, Ghrelin and their receptors, as well as the downstream signaling molecules include Ca2+、PKA、PKC、MLC etc. So we can further clarify the mechanism of gastrointestinal motility disorders in anorexia and the mechanism of the tool drug efficacy, and hope to preliminary probe the material basis of the containing serum pharmacological effects. The study not only provides new research ideas and advanced experimental methods, but also can fill in the gaps of traditional Chinese medicine research in this field, so that we can more effectively guide and evaluate the clinical treatment.

本项目在建立运脾开胃法治疗厌食症的深层疗效机理可能与干预VIP、Ghrelin信号通路有关的科学假说基础上，针对国际有关消化系统新的研究热点（胃肠肽受体、信号接受、信息传导等），克服以往对厌食症疗效机理的研究多限于初始信号分子量的异常与终端效应器功能障碍等，着重于过程中信号传导等中间环节。运用Real-Time PCR、激光共聚焦、膜片钳等多种先进技术，观测运脾开胃治法工具药-运脾开胃颗粒对胃肠激素VIP、VIPR与Ghrelin、GhrelinR，及Ca2+、PKA、PKC、MLC等下游信号通路的影响，深入阐明厌食症胃肠动力紊乱发生机制及工具药疗效机理，并初步探明其血清药理效应物质基础。该研究不仅提供新的研究思路与先进的实验手段，且可填补中医药在相关领域的空白，从而更有效地指导与评价临床治疗。

本项目在建立运脾开胃法治疗厌食症的深层疗效机理可能与干预VIP、Ghrelin信号通路有关的科学假说基础上，运用Real-Time PCR、激光共聚焦、膜片钳等多种先进技术，观测运脾开胃治法工具药-运脾开胃颗粒对胃肠激素VIP、VIPR与Ghrelin、GhrelinR，及Ca2+、PKA、PKC、MLC等下游信号通路的影响，深入阐明厌食症胃肠动力紊乱发生机制及工具药疗效机理，并初步探明其血清药理效应物质基础。.通过构建厌食症动物模型，观察不同剂量运脾开胃方对进食量、体重的影响，明确该方治疗厌食症的效应。从整体动物及细胞水平研究工具药与VIP、Ghrelin信号通路的关系，并通过拮抗信号通路关键信号，如Ghrelin受体、Ca2+、VIP受体等，反复验证起效机理。研究结果表明：①在整体动物水平，运脾开胃方可有效改善厌食症大鼠模型出现的进食量减少及体重下降，显著上调厌食症大鼠血清和胃窦部Ghrelin的表达，对胃窦、空肠部的p-IP3R、p-PKC、p-MLC亦有不同程度的上调作用，能不同程度上调胃窦、空肠部VIP信号通路下游蛋白p-PKA、p-eNOS的表达水平。这些变化在干扰信号通路后出现不同程度改变，说明运脾开胃方治疗厌食症的机理与Ghrelin及VIP信号通路密切相关。②在细胞水平，运脾开胃方含药血清能够显著降低人胃窦平滑肌细胞(HGSMC)中VIP及其受体 mRNA表达量，下调VIP受体蛋白表达，该结果与动物实验不符，推测可能该方对该信号系统的作用不具有特异性及稳定性。同时， Ghrelin mRNA表达下降与动物实验不符，但在GHSR mRNA、 p-IP3R、p-PKC、p-MLC的表达方面，与动物实验一致，这一结果可能与我们采用的是HGSMC作为细胞模型有关，因其并未出现厌食症相关胃肠肽表达的变化，但仍可以推论该方可对Ghrelin信号通路产生作用，体内体外实验具有一致性，其机制可能与激活Ghrelin受体GHSR促进细胞内Ca2+释放有关。③运脾开胃方的水提物中检测出12个化学单体，可能为运脾开胃方的物质基础，其中苍术素可激活HGSMC表面的GHSR，促进Ca2+内流，进而上调细胞内p-MLC水平，同时经由GHSR促进小鼠胃排空。运脾开胃复方治疗厌食症大鼠的机理可能与调节VIP、Ghrelin信号通路、改善胃肠肽表达、促进胃肠动力有关。